BiomoleculeâMercury Interactions: Modalities of DNA BaseâMercury

Chem. Rev. 2004, 104, 5911−5929

5911

Biomolecule−Mercury Interactions: Modalities of DNA Base−Mercury Binding Mechanisms. Remediation Strategies Ikenna Onyido,*,† Albert R. Norris,‡ and Erwin Buncel*,‡ Department of Chemistry and Center for Agrochemical Technology, University of Agriculture, Makurdi, Nigeria, and Department of Chemistry, Queen’s University, Kingston, Canada K7L 3N6 Received September 15, 2003

Contents 1. Introduction 2. Modalities for Binding of Mercury to Biomolecules 3. Complexes with Nucleosides and Related Substrates 3.1. Adenine Derivatives 3.2. Guanosine and Inosine Nucleosides and Related Compounds 3.3. Theophylline and Xanthosine Nucleosides 3.4. Imidazole, 1-Methylimidazole, and 1,3-Dimethylimidazolium Ion 3.5. Sulfur-Modified Nucleosides and Related Substrates 3.6. 7-Methylguanine, a Minor t-RNA Base 3.7. Nucleotides and Related Substrates 4. Complexes with Amino Acids and Derivatives 4.1. Cysteine 4.2. Gluthathione 4.3. Penicillamine 4.4. Other Amino Acids 5. Competition and Exchange Reactions: Probes for the DNA Binding Mechanism 6. Diagnostic and Practical Utility of NMR Features of CH3HgII Complexes 7. Neurotoxicity of CH3HgII 8. Mercury Detoxification Strategies 8.1. Anthropogenic Methods 8.2. Nature’s Strategies 9. Concluding Remarks and Future Outlook 10. Acknowledgments 11. References

5911 5912 5913 5913 5914 5915 5916 5917 5918 5918 5919 5919 5919 5919 5920 5920 5922 5924 5924 5924 5925 5926 5927 5927

1. Introduction The evident ubiquity of heterocyclic residues in nature as key constituents of many biologically important molecules such as proteins, enzymes, vitamins, nucleic acids, etc. and the demonstrated requirement for metal ions for a variety of physiological processes in plants and animals1 provide the impetus for investigations into metal ion-biomolecule interactions. As well, metal ions have been shown to have far-reaching biological and environmental consequences.1,2 These investigations are † ‡

University of Agriculture. Queen’s University.

diverse in nature, involving a variety of metal ions and a range of simple to complex heterocyclic residues of key biological molecules. Binding of metal ions to the heteroatomic sites of biomolecules is without doubt fundamental to their observed physiological effects.1,3 Considerable attention has therefore been attached to understanding the structural, kinetic, and thermodynamic details of these interactions in a number of laboratories, as a key requirement for unraveling and discussing the mechanisms of action and physiological roles of metal ions in living systems. Research in this domain in our laboratory spanning over two decades has focused largely on two aspects. The interactions of the heavy metal mercury, mainly as CH3HgII, with DNA bases and other model systems have been studied as a probe of the molecular basis for its toxicity and environmental effects. Structural features of these complexes as revealed through spectroscopic studies bear relevance to the biological chemistry of HgII. Novel complexes have been isolated in a number of cases, and these provide insight into additional pathways for the physiological action of metals and clues for remedial intervention. The second aspect of our work concerns isotopic hydrogen exchange in heterocycles such as imidazole, histidine, and thiazole complexed to metal ions, deriving from the fact that isotopic hydrogen exchange of biomolecules in different environments has been a practical tool for probing biological function.4 These studies have been expanded to include metal ions other than CH3HgII, to investigate substitution-inert complexes5 and to explore the chemistry of PtII-based complexes as anticancer agents.6 These studies7-9 have yielded significant information on the relative catalytic abilities of metal ions as surrogates for the proton,10a and emphasize the importance of the electronic structures of metal ions in determining reactivities in the ligand portion of the metal complexes. An account of our studies on metal ion effects on isotopic hydrogen exchange in imidazoles and related substrates has been published recently.10b Sigel and co-workers11 have recently quantified the relative acidifying effects of protonation and metalation in a number of DNA bases and related substrates. The above two aspects of our research have a functional linkage, to the extent that structurereactivity relationships and electronic effects highlighted by the dynamics of isotopic hydrogen exchange enable an evaluation of factors responsible

10.1021/cr030443w CCC: $48.50 © 2004 American Chemical Society Published on Web 10/06/2004

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Ikenna Onyido graduated from the University of Ibadan, Nigeria, with a B.Sc. First Class Honors (1974) and Ph.D. (1979) with a thesis on mechanisms of aromatic nucleophilic substitution under the supervision of Jack Hirst. After a year of postdoctoral work with Per Ahlberg at the University of Uppsala, Sweden, he returned to Ibadan to take up a lectureship position in chemistry. In 1989, he was appointed Professor and Head of the Department of Chemistry in the new University of Agriculture, Makurdi, where he also served as the foundation Dean of Science and, later, became Deputy Vice-Chancellor for four years and Acting Vice-Chancellor for one year. He is currently the Director of the Center for Agrochemical Technology in Makurdi, which is concerned with the applications of chemical science and technology for the realization of sustainable agriculture. Since 1983, Ikenna has maintained an active collaboration with Erwin Buncel, with whom he shares common research interests in different aspects of chemistry.

Albert R. Norris, born in Meadow Lake, Saskatchewan, Canada, in 1937, earned B.E. (chemical engineering) and M.Sc. (chemistry) degrees at the University of Saskatchewan in 1958 and 1959, respectively. He obtained his Ph.D. in chemistry at the University of Chicago in 1962 for research carried out under the supervision of Professor Weldon G. Brown. From 1962 to 1964, an NRC Fellowship enabled him to do postdoctoral research at University College, London, under the direction of Sir Ronald Nyholm and Dr. Martin Tobe. He was appointed an Assistant Professor of Chemistry at Queen’s University in 1964 and became a full Professor in 1976. He was made a Fellow of the Chemical Institute of Canada in 1977. Over the years he has maintained a keen interest in the unique Engineering Chemistry program at Queen’s and served as Undergraduate Chair of the program from 1990 to 1998. He was designated a Professional Engineer in the province of Ontario in 1989. His main research interests have been in the areas of σ and π complexes of nitroaromatic compounds, the oxidation and reduction reactions of coordinated ligands in inert transition-metal-ion-containing complexes, and metal ion−biomolecule interactions. He has been an Emeritus Professor at Queen’s since 1999.

for binding site preferences and selectivities noted in the structural studies of metal ion-biomolecule interactions. A number of metals such as Pb, As, Bi, etc. have proven toxicity and deleterious environmental con-

Onyido et al.

Erwin Buncel, Professor Emeritus of Chemistry at Queen’s University, is a physical organic chemist with strong interests in bioorganic/bioinorganic chemistry (for a previous biographical account, see Chem. Rev. 1995, 95, 2261). Recent research in Buncel’s group in this area has focused on metal ion−biomolecule interactions with an environmental emphasis. Other major interests include photochromic materials, molecular switches, and carbanion chemistry. Buncel is the author/coauthor of 300 research papers in these areas as well as a number of chapters/review articles and monographs. He was the recipient of the SYNTAX Award in Physical Organic Chemistry in 1985 and the R. U. Lemieux Award in Organic Chemistry in 1999, both from the Canadian Society of Chemistry. He was an editor for the Canadian Journal of Chemistry (1981−1993) and for the Journal of Labelled Compounds and Pharmaceuticals (1995−2002). As an Emeritus Professor, Erwin is continuing an active research program with graduate students, postdoctoral fellows, and colleagues in different countries, and looks to continuing challenges that the world of chemistry holds.

sequences. We have focused mainly on Hg because of its wide distribution in the environment as organomercurials, mainly CH3HgII, through an elaborate number of chemical and biological pathways.12,13 Over time, the anthropogenic activities of our industrial society have resulted in a significant load of mercury in the biosphere, leading to widespread contamination of water and soils with attendant environmental and health concerns. This review elucidates the various modes of mercury interaction with DNA bases and other biologically important molecules, identifying, in each case, the binding sites of the substrates of interest. The relevance of competition and exchange reactions between HgII/CH3HgII and nucleobases to the mechanism of DNA binding with mercury are explored. In addition to discussing the diversity of structural types encountered in CH3HgII/HgII complexes of biomolecules and the mechanism of HgII binding to DNA, this review also highlights recent work on mercury detoxification strategies, encompassing anthropogenic and nature-based methods. The necessity for exploring chemical mimics of nature in addressing the issues of environmental remediation of mercury pollution and mercury intoxication in humans is emphasized.

2. Modalities for Binding of Mercury to Biomolecules The binding of heavy metals to DNA results in potential toxic effects.14 The pattern of HgII binding has been used to probe DNA structures in viruses and nucleosomes, as well as to separate DNAs of

Biomolecule−Mercury Interactions

different base compositions.14,15 CH3HgII is one of the most toxic forms of HgII; its environmental significance has been underscored by studies which reveal the chemical and microbiological transformations of HgII into CH3HgII and its bioaccumulation.16 Being a prototype soft acid which shows strong preference for unifunctionality and minimum steric effects, it has been used as a selective probe for unpaired bases in superhelical DNA14,17 and has shown greater tendency than HgII to partition into lipids or hydrophobic regions of the cell.18 Chronic intake of CH3HgII at subtoxic levels results in chromosomonal damage in humans, presumably due to its direct interaction with DNA; the mutagenic nature of organomercurials in general has been demonstrated19 and is now well recognized. Inasmuch as the interaction of metal ions with the sulfur atoms of nucleosides and amino acids bearing the thiol group has provided the dominant mechanism for explaining the deleterious biological effects of heavy metals,20 certain aspects of metal ion toxicity, e.g., mutagenic effects, may not be satisfactorily explained by solely invoking binding to DNA or protein sulfur functions.21 Ribose/ribophosphate groups and purine/pyrimidine bases which abound in biomolecules present several N and O donor atoms as potential binding sites.22 Binding has been demonstrated for copper and uranyl ions to the ribose moiety in nucleobases, in addition to coordination of metal ions to endocyclic and exocyclic N sites of DNA bases.3b Metal ion ligation to nucleobases is thought to give rise to nucleobase mispairing; this phenomenon has been suggested as being relevant to the mutagenic potential of metal ions6a and the altered sequence of amino acids in proteins resulting from interference with the transmission of genetic information in protein synthesis.2a Our studies sought to provide an insight into the modes of heavy metal binding to nucleic acids and other biomolecules, with a view to assessing the importance of N and O donor atoms in contributing to the overall effects of heavy metal ions. Structural evidence for the isolated complexes was obtained primarily from 1H/13C as well as 199Hg NMR studies, X-ray, and IR spectroscopy. It is important to note that although O atoms in the sugar portion and/or the carbonyl function(s) of the substrates investigated are possible binding sites, no definitive evidence was found for binding of HgII/CH3HgII at these sites in nucleobases. The variety of structures and bonding patterns realized in these studies enrich our understanding at the molecular level of metal ion effects in biological systems and provide additional pathways for discussing DNA base-HgII/CH3HgII binding mechanisms and the effects of HgII/CH3HgII coordination on the secondary structures of DNA. As shown in the sections following, HgII/CH3HgII complexes of DNA bases with metal:ligand ratios of 1:1, 2:1, and 3:1 have been reported. Coordination to N1, N3, N7, N9, and exocyclic NH2 in these nucleobases has been demonstrated. In addition, binding of metal ions to C8 has been realized under H+ or metal ion activation at N7. Bridged species of the types Nuc1-Hg-Nuc1, Nuc1-Hg-Nuc2, and Nuc2-

Chemical Reviews, 2004, Vol. 104, No. 12 5913

Hg-Nuc2 have been characterized. S-ligation of HgII/ CH3HgII is a facile process with these biomolecules whenever the thiol group is part of the ligand structure. Amino acids such as cysteine, penicillamine, glutathione, etc. exhibit NH3+ coordination; amino acid COO--electrophile binding has been shown to occur in the solid state, but not in solution. Thus, there is an inherent diversity of coordination patterns in these interactions, whose binding modalities have been elucidated with spectroscopic methods and in which X-ray diffraction has played a significant role.

3. Complexes with Nucleosides and Related Substrates 3.1. Adenine Derivatives Beauchamp and co-workers23 showed that adenine reacts with CH3HgII at pH 9 and r ) 1 (r is defined here and in all cases as the ratio [metal ion]/[ligand]) to yield 1. With r ) 2 under neutral conditions or in the presence of 1 equiv of NaOH relative to adenine, the product of the interaction is 2.23b With r ) 3 and 1 molar equiv of NaOH or r ) 4 without added NaOH, a 3:1 metal-base complex with structure 3 is obtained. Complex 4a is formed at r ) 4 when the pH is adjusted to 7 with NaOH.23b The structure of 4a was initially deduced from IR spectra23b,d and subsequently solved by X-ray crystallography.23e In H2O-EtOH mixtures,24 the amino group is doubly metalated to yield 4b. Significantly, no binding of CH3HgII to N1 was observed with adenine in these systems, contrasting the behavior of adenosine and 9-methyladenine (vide infra).

N6,N6-Dialkylaminopurines25 yield complexes analogous to 1 at r ) 1; in the presence of 1 M CH3HgOH, a 2:1 complex of type 5 with metal binding to N3 and N9 resulted. Using high metal-to-ligand ratios gives a new complex, 6, in which N7 is now protonated. These results highlight the steric influence of the alkyl groups at N6 on the complexation process.


The reaction of a slight excess of 9-methyladenine (9-MeAd) with ethanolic trismethylmercurioxonium gave 7a, as a mixture of syn and anti isomers,26 favoring the former presumably due to steric factors. A ribose analogue of 7a was obtained when adenosine (AdoH) was reacted with a slight excess of the organomercurial hydroxide.26 With 2 equiv of CH3HgOH and 1 equiv of 9-MeAd in CH3CN or DMF, a neutral compound, 7b, in which both NH2 protons have been replaced by linearly coordinated CH3HgII, results.27 In the presence of 1 M NaOH and with r ) 2, 9-MeAd yields a 2:1 complex in which CH3HgII is attached to N1 and N6, i.e., 8, existing mainly in its anti conformation.23a On the other hand, N7 is the primary binding site for 3-methyladenine (3-MeAd),28a which has been shown29 by semiempirical calculations and NMR spectroscopy to exhibit thermodynamic preference for its amino form. 3-MeAd gave, respectively, 9 at r ) 1 and pH 4, 10 at r ) 1 and pH 2, and 11 at r ) 3 and pH 4. NMR evidence suggests28a that complexes 12 (r ) 1) and 13 (r ) 3) were also obtained at pH 8. For the structures of 9-13, NMR data indicate significant reduction in electron density in the imidazole ring, in accord with the structures shown.

8-Azaadenine28b forms a 1:1 complex analogous to 1 at r ) 1 and pH 4; at pH 2, N1 is protonated to yield 14. With r ) 2 and pH 5, a complex in which CH3HgII coordinates to N3 and N9 results, while at r ) 3 and pH 6.5 methylmercuration of N1, N6, and N9 occurs to give 15. Blocking N9 in 8-azazadenine through methyl substitution alters28c its binding pattern to give, at r ) 1 and pH 2-3, a complex which has structure 16 in the solid state; in DMSO-d6 solution, NMR evidence suggests structure 17 as the product of this interaction. With r ) 2 and pH ≈ 5,

Onyido et al.

coordination occurs at N1 and N6 of 9-methyl-8azaadenine to give 18, believed to exist in two isomeric forms. With r ) 5 and pH g 7, a complex analogous to 7b was obtained. It is important to note that, with these aza-modified adenines, no complex was obtained with CH3HgII binding at N7 or N8.

3.2. Guanosine and Inosine Nucleosides and Related Compounds A number of complexes of guanosine (GuoH), 19a, and inosine (lnoH), 19b, with HgII and CH3HgII in solution were postulated by Simpson30 on the basis of UV studies, and confirmed through Raman spectroscopy by Tobias and co-workers.31 With r ) 1 at low pH, N7-coordination of the electrophile to these nucleobases results in 20a and 20b, while reaction at high pH affords the N1-bound products 21a and 21b, isolated as solids.32 In addition, 22a and 22b were reportedly obtained at high and low pH, with r ) 2. Our work33,34 not only reproduced the isolation and characterization of 20a, 20b, 21a, and 21b but also revealed additional features of the interaction, to include the formation of 3:1 CH3HgII-nucleoside complexes. With GuoH and InoH, at pH 3 and r ) 2, 22a and 22b were obtained, respectively; at pH 7, an additional product characterized as 24 was obtained in each case in low yield, on leaving the reaction mixture for 48 h. Complex 24b was also obtained in reasonable yield at pH 7 and r ) 3 by heating the reaction solution to 50 °C; a similar situation obtains with GuoH, although incomplete substitution of C8-H by CH3HgII results due to an additional interaction of the electrophile with the exocyclic NH2.33 The highlight of this study was the first reported isolation of a purine nucleoside C8HgII bonded complex, 24; the greater stability of 24 relative to its N-bound counterpart was unambiguously demonstrated by the 2J(1H-199Hg) values: 159.5 and 215.8 Hz for C-HgIICH3 and N-HgIICH3, respectively. Formation of 24 results from deprotonation of 22 to generate the resonance-stabilized ylide intermediate 23 at pH 7. Proton abstraction from 22 by HO- is rendered facile by the activating influence of the N7-coordinated electrophile. The irreversible addition of CH3Hg+ to 23 essentially precludes its reprotonation by H2O as occurs in hydrogen exchange processes.7-9



Scheme 1

Scheme 1 summarizes the range of structures obtained with GuoH and InoH under the conditions of pH and stoichiometries indicated. The essentially irreversible formation of 24 and the thermodynamic preference of C8-bound CH3HgII complexes over their N1- and N7-bound counterparts suggest an alternative and plausible mechanism for the observed mutagenicity of organomercurials. Binding at N1 and the exocyclic NH2 group could have implications for the disruption of base-pairing capabilities of GuoH and InoH, with far reaching consequences, especially for the secondary structures of polymeric biomolecules. Methylmercuration of the exocyclic NH2 group has been demonstrated26 in GuoH, 19a, 1-methylguanosine (1-MeGuo), 19c, and cytidine (Cyt), 25a, as well as in 9-MeAd and AdoH

described above. Clarke35 had earlier adduced spectroscopic and chemical evidence for binding of RuIII to deprotonated NH2 groups in adenosine, cytidine, and tubercidine. A 2:1 CH3HgII-Guo adduct was obtained26 with 19a, consistent with CH3Hg+ binding at N1 and deprotonated NH2. With a slight excess of CH3HgOH over substrate, 1H NMR spectra of isolated complexes revealed the NH2 group also as the primary target in 19c and 25a. Since N-HgIICH3 bonds of exocyclic nitrogens are stronger than those of their endocyclic counterparts,36 HgII/CH3HgII binding to exocyclic NH2 groups is potentially capable of causing greater disruption to base pairing than the labile methylmercuration of endocyclic thymine N3 or guanine N1 positions, from a thermodynamic perspective.

3.3. Theophylline and Xanthosine Nucleosides Theophylline (ThH), 26, and xanthosine (XanthH2), 27, exhibit a number of potential binding sites and are considered valid models for certain DNA bases and their derivatives.36 Metal binding to 26 occurs through deprotonation of N7-H, since methyl substitution precludes reaction at N1 and N3; a neutral


N7-bound metal complex, realized through the displacement of N7-H by the electrophile, has been described for this compound.37 Our study38 shows that initial CH3HgII binding in 26 is pH-dependent, giving a range of products in which coordination occurs also at N7 and N9 at r > 1. At pH 8-9 and r ) 1, 28 is the product which has been unambiguously shown by X-ray analysis39 to bind the electrophile at N7. The crystal structure of the monohydrate of 28 shows the roughly linear N7-coordination to CH3HgII with the expected values of bond lengths for Hg-N7 ) 2.06 Å and Hg-CH3 ) 2.04 Å. Hg‚‚‚O(6) bonding in 28 is probably very weak or absent, judging from its distance (3.18 Å); an intermolecular contact of 2.98 Å is established between N9 and Hg. The H2O molecule forms a moderately strong Hg-O bond (2.94 Å) to Hg and is simultaneously H-bonded with carbonyl C2-O2 and C6-O6 of two different molecules.

Onyido et al.

exchange of the CH3HgII moieties between these positions. Reaction of 26 with Hg(OAc)2 leads to the formation of 30, which demonstrates HgII bridging between two deprotonated N7 sites; protonation of 30 in acidic aqueous solution yields 31. Treatment of 30 with an additional molar equivalent of CH3HgII at pH 2-3 affords 32, which can also be accessed directly from 31 by reacting the latter with another equivalent of the electrophile under basic conditions. Significantly, no C8-bound HgII/CH3HgII complexes are realized with 26, showing clearly that the two electrophilic groups at N7 and N9 in 29b have not provided sufficient activation for C8-H abstraction, contrasting the behavior of inosine33,34 discussed above and xanthosine (see below). Xanthosine, 27, has two ionizable protons in the pyrimidine moiety, making it a good model for uracil and thymine as pyrimidine-type nucleic acid constituents. Treatment of 27 with CH3HgOAc at pH 5 and r ) 1 afforded 33a in good yield,40 consistent with the order of acidity N3-H > N1-H.41 At pH 8, 33a was formed along with small quantities of 33b as shown by 1H and 13C NMR, due to the disproportionation reaction depicted in eq 1. Similar dispropor-

33a + 33a h 33b + 27

(1)

tionation reactions have been observed in the CH3HgII/imidazole (see below) and CH3HgII/adenine23a systems. Pure 33b was obtained at pH 8-9 with r ) 2; further treatment of 33b with 1 equiv of CH3HgII yields 34b, which at pH 5-6 undergoes C8-methylmercuration with a further equivalent of the electrophile to form 35. Under acidic conditions (pH 2-3)

and with r ) 1, 27 yields 34a in which CH3HgII coordinates to N7. Thus, depending on the pH and reactant stoichiometry, methylmercuration of xanthosine is possible at all four potential sites: N1, N3, N7, and C8. The dichotomy between xanthosine and theophylline in terms of the occurrence of C8-methylmercuration in the former but not in the latter is worthy of note.

3.4. Imidazole, 1-Methylimidazole, and 1,3-Dimethylimidazolium Ion

Treatment of 28 with 1 M HNO3 (pH 2-3) yields 29a, which bears H+ at N9; 29a can also be accessed directly from equimolar mixtures of 26 and CH3HgII at pH 2-3. Reaction of 28 under neutral conditions or 29a under basic conditions with another equivalent of CH3HgII affords 29b in which CH3HgII is coordinated to N7 and N9; NMR data indicate rapid

Potential binding sites decrease progressively in the series42 imidazole, 36a, 1-methylimidazole, 36b, and 1,3-dimethylimidazolium ion, 37. A 1:1 molar mixture of 36a and CH3HgNO3 in H2O yields 38a; an ethanolic solution of 36a reacts with aqueous CH3HgNO3 to form 36c, while 38b, the product of methylmercuration at both N1 and N3 according to the disproportionation reaction of eq 2, is obtained

38a + 36c h 38b + 36a

(2)

under neutral or basic conditions.42a Similar results



for 36a were reported by Rabenstein and coworkers.42b,43 Precedents for eq 2 exist in the adenine/ CH3HgII 23a and xanthosine/CH3HgII 38 systems (vide supra). The symmetrical Hg-bridged products 39a and 39b are obtained when 36a and its 4-nitro derivative are treated with ethanolic HgO. Structures of complexes of 4-nitroimidazole with CH3HgII and AgI have been described.42c

1-Methylimidazole, 36b, bears close resemblance to the five-membered ring portion of the purine nucleosides 19a, 19b, and 27, already studied.33,34 An equimolar mixture of 36b and CH3HgNO3 affords the N3-bonded species 38c. At pH 7 and r ) 2, the crude product realized was shown by NMR to be a mixture of 38d and 40a. Subsequent recrystallization of the crude product from hot water yields 40a only, suggesting a symmetrization process according to eq 3.

38d + 38d h 40a

(3)

No methylmercuration occurs with 37 at low pH; at high pH 38e was observed in solution, consistent with C2-H/C8-H bond activation being prerequisite for C2/C8 methylmercuration33,34 (see Scheme 1). Attempted recrystallization of 38e from H2O leads to isolation of the symmetrized product 40b (cf. eq 3 for formation of 40a).

C2-binding by HgII/CH3HgII was established for 36b under conditions for N3 protonation and for 37, but not for imidazole, 36a. The formal difference is methyl substitution at N1 in 36b and 37 but not in 36a. Similarly, C8-methylmercuration occurs in 19a, 19b, and 27, all of which bear the ribosyl group at N9, but not in 26, where there is a proton on N9.33,34,38

This difference has been explained on the basis of the concept of the “minimum degree of activation” requirement.42a

3.5. Sulfur-Modified Nucleosides and Related Substrates Complex formation has been investigated between CH3HgII and the following S-modified substrates: 1-methylimidazoline-2-thione, also called methimazole (MeImSH), 41/42,44 8-thioguanine (8-thioGuoH2), 43,45 and 6-mercaptopurine riboside (6-MPurH2), 44a.46 MeImSH exists in tautomeric equilibrium47 as

shown in eq 4, whereas 43 exists predominantly as the thione form in solution and in the solid state. The biological importance and practical utility of these substrates, which incorporate the soft S atom in their structure, have been variously described.48 KT

41 y\z 42

(4)

Reaction of 41/42 with CH3HgII affords44 the Sbound complexes 45 and 46a at high and low pH, respectively, with r ) 1, contrasting a literature report49 of binding of PdII and PtII to this substrate via N3 at low pH and via both N3 and S at high pH. With r ) 2, further replacement of N3-H occurs in 46a to give 46b. Thus, the introduction of the S atom to the parent compound 36b shifts the primary reaction center from N3 to S, in accord with the documented preference of HgII for soft donor atoms.50 The ability of this ligand to compete for HgII with S-containing bioligands makes it a potential protective agent against HgII intoxication. Thiazolidine-2thione complexes, analogous to 45, 46a, and 46b, have been reported.51

Unambiguous information regarding binding sites in 45 and 46a was obtained by X-ray structure


analysis,52 which confirmed conclusions on structural features reached on the basis of chemical and spectroscopic data44 and revealed the following details: H+ is bound to N3 while CH3HgII is attached to sulfur. Mercury in the complexes exhibits linear coordination, with S-Hg-C6 ) 176.1°; the Hg-C bond is a normal one (2.09 Å). The exocyclic Hg-S bond (2.382 Å) is typical of 2-coordinated Hg, forming a number of secondary bonds with NO3 but not coplanar with the ring (Hg-S-C2-N1 ) -134°, with the angle at S ∼100°). H-bonds are formed between NO3- oxygens and the acidic proton on N3. The Hg-S bond in 45 (2.338 Å) is significantly shorter than in 46a (2.382 Å), probably due to the absence of intermolecular contact between Hg and donor atoms on adjacent molecules. In both 45 and 46a, the C2dS bond in the parent compound (1.691 Å) is lengthened upon coordination with CH3HgII, suggesting that it approximates a single bond in the complexes. Site preferences in CH3HgII binding with 43 are both pH- and stoichiometry- dependent.45 At pH 8-9 and r ) 1, the S-bonded complex 47a results; also at this pH and when r ) 2, 47b, which manifests N1binding in addition to S-methylmercuration, is obtained. Under acidic conditions (pH 1-2), the ionic S-bonded product 48a is obtained at r ) 1. Reacting 47a with a further mole equivalent of CH3HgII yields the cationic product 48b in which methylmercuration has occurred at N9. A 3:1 cationic complex, 48c, is realized by treating 47b with an additional equivalent of CH3HgII at pH 2-3.

Onyido et al.

HgII-nucleoside complexes 50c and 50d, in which there is C8-H displacement at pH 7-8. Reaction of 44a and 44b under acidic conditions (pH 2-3) and with r ) 1 affords the cationic products 51a and 51b. Thus, S-methymercuration is achieved first, before N- and/or C-methylmercuration occurs in all cases, formation of the latter species being dependent on the pH and reactant stoichiometry. Furthermore, the occurrence of C-methylmercuration accords with our finding on C8-H/C2-H activation in inosine, xanthosine, and imidazole derivatives, resulting from CH3HgII coordination to N7/N3 in these systems, which facilitates proton abstraction and ylide formation.26,33,42a

3.6. 7-Methylguanine, a Minor t-RNA Base

In relation to the parent substrates guanosine (19a) and inosine (19b), already investigated,33,34 in which N1, N7, and C8 were found to be binding sites, the mercapto analogues 44a and 44b contain the S atom as a likely additional coordination site.46 A range of products is possible depending on the pH of the medium, reactant stoichiometry, and relative affinities of N, S, and C centers toward CH3HgII.47 At pH 7-8, reaction occurs at the exocyclic S of the pyrimidine moiety in both 44a and 44b46 to give 49a and 49b, respectively; further reaction with 1 molar equiv of CH3HgII at pH 2-3 affords 50a and 50b, respectively, in which N7-methylmercuration is realized. These products are converted to the 3:1 CH3-

Binding of CH3HgII to the minor t-RNA base 7-methylguanine (7-MeGua) was studied by Sheldrick.53 Two different 1:1 complexes with CH3HgII coordination at N1 and N9 were obtained at r ) 1 in the pH ranges of 9-12 and 1-4, respectively. With r ) 3, a 2:1 complex in which the metal is attached to N1 and N9 was isolated in the pH range 4-6; a change of the pH range to 1-3 gave a 3:1 complex with metal bonding to N1, N3, and N9. No evidence was found for metal bonding to N7 and the exocyclic NH2. 9-Methylguanine (9-MeGua), on the other hand, gave solid complexes analogous to 20a, 21a, and 22a; binding to N3 was not observed.54

3.7. Nucleotides and Related Substrates The results presented above do not reveal any interactions of HgII and CH3HgII with the O donor atoms of the sugar and phosphate moieties in nucleosides and their analogues. This observation is relevant in the following discussion of recent literature on the interaction of these electrophiles with nucle-


otides and related substrates. Section 5 below touches on the structural and conformational consequences of binding interactions involving polynucleotides. 1 H NMR spectroscopic study of the interaction of d[CGCGAATTCGCG]2 with HgII reveals decreasing intensities of the thymine imino protons with increasing metal ion concentration and a concurrent downfield shift of the guanine G4 imino hydrogens.14,55 Decreasing thymine proton signals are consistent with N3-HgII binding, following deprotonation of imino hydrogen. Alternatively, this could be a result of fast exchange of the thymine imino protons with the aqueous environment, consequent upon HgII binding to other adenine and thymine sites. Earlier studies with nucleases demonstrate preferential binding of HgII to thymine whereas, CuII favors guanine.56 The opposite effects of HgII and CH3HgII on staphylococcal nuclease digestion of calf thalamus DNA, whereby the DNA cleavage rate is increased irreversibly by CH3HgII but decreased reversibly by HgII, indicate that the secondary structure of DNA is unaffected by HgII.57 On the other hand, CH3HgII breaks down the secondary structure of DNA to give single strands which are rapidly and irreversibly hydrolyzed by staphylococcal nuclease.58 In a recent study,59 two simple mononucleotide models of DNA, 2′-deoxyguanosine 5′-methylmonophosphate (MepdG) and 2′-deoxyguanosine 3′-methylmonophosphate (dGpMe) were used to demonstrate strong binding affinity of HgII for guanosine N7. High-field multinuclear (1H, 13C, 15N, and 31P) NMR evidence was obtained for the strengthening of the anomeric effect through binding of HgII to N7, leading to an attenuation of the electron density in the imidazole moiety.

4. Complexes with Amino Acids and Derivatives 4.1. Cysteine CH3HgII coordinates with cysteine over the pH range 0-14 to form a 1:1 complex.60 Of the three potential binding sites in the molecule, only the sulfhydryl group is involved in the 1:1 interaction, consistent with the observation that no binding is observed with S-methylcysteine. X-ray crystallographic studies60b,c have shown that the electrophile is bound to the deprotonated sulfhydryl group, and that a weak intramolecular Hg‚‚‚O interaction (2.85 Å) exists with the carboxylate group. Replacement of the S with Se in cysteine gives a complex with a stronger metal-ligand bond;61 this may account for the protective effects against CH3HgII poisoning ascribed to selenium-based compounds.14 In vivo formation of a CH3HgII-cysteinylglycine complex has been reported.14 By contrast, methionine is coordinated to CH3HgII via the amino nitrogen.62 The formation of the 2:1 complex 52 at high pH and in the presence of excess metal ion has been reported;63 52 manifests CH3HgII coordination to S and N sites. Potentiometric evidence has been presented63 for the formation at low pH of a 2:1 complex analogous to 53 (see below) in which the two CH3HgII moieties are bound to sulfur.


4.2. Gluthathione Gluthathione (GSH) is a physiologically important sulfhydryl-containing tripeptide. As the predominant low molecular thiol in all living organisms, it has been studied as a model compound for the binding of mercury to S-amino acid residues of proteins. In the reduced form, this peptide has been shown to attenuate the cytotoxic effects of mercury.14 GSH has been implicated in a host of cellular biochemical processes,64 including the protection of the living cell against free radicals,65 oxidation damage,66 thermosensitivity,67 and sulfhydryl-reactive agents.68 The roles of GSH in the protection of bacterial cells and higher life forms against heavy metals and xenobiotic toxicology have been discussed.69,70 GSH has been identified by 1H NMR as a major binding site for HgII in intact human erythrocytes71 and has been implicated in several other studies of HgII toxicology.72 CH3HgII coordinates to GSH over the pH range 0.5-13 in a manner that is pH-dependent.60 At pH < 4, a 2:1 complex is formed in which two CH3HgII cations are attached to the sulfhydryl group, resulting in the charged complex 53; between pH 4 and pH 8, there is a shift of one CH3HgII to the amino group. Dissociation of the second CH3HgII occurs at pH 10 and above, presumably to form CH3HgOH. A Raman spectroscopic study73 of the CH3HgII/GSH system revealed that, at r ) 1, a 1:1 complex was formed via coordination of the eletrophile to S; a value of log K ) 15.9 has been measured for the formation constant of the 1:1 complex.43 Methylmercuration of the sulfhydryl group is the favored step in CH3HgII-GSH interaction. Complexes of the types Hg(glutathione)274 and Hg(glutathione)375 with formation constants log K(25 °C) ) 40.9574 and 3.28,75a respectively, were obtained at physiological pH and r < 0.5. Although binding of the third ligand to form Hg(glutathione)3 is much weaker than binding of the two ligands in Hg(glutathione)2, it is sufficiently strong to ensure that a significant fraction of HgII is present as Hg(glutathione)3 at physiological pH and excess metal ion (i.e., r < 0.5).75 The multiplicity of potential GSH-HgII complexes and the favorable thermodynamics for their formation are indicative of the possible role of glutathione in mercury remediation (see below).

4.3. Penicillamine A 1:1 S-bound complex with CH3HgII was reported by Rabenstein and Fairhurst60a for the sulfur amino


acid penicillamine. The amino acid moiety was shown to be present in the complex in its zwitterionic form and coordinated to the metal via a deprotonated sulfhydryl group.76 The 2:1 complex CH3HgSC(CH3)2CH(NH2CH3Hg)COO- (54), isolated by Carty and co-

workers76 from an aqueous solution, exhibits bonding of CH3HgII to the deprotonated S and N centers, as shown by X-ray analysis. C-Hg-S and C-Hg-N bonds deviate from linearity possibly due to Hg interactions with neighboring S and O atoms. Phenylmercury(II) complexes of penicillamine analogous to those of CH3HgII have also been reported;77 these decompose to form diphenylmercury when stirred as suspensions in benzene at ambient temperature.

Onyido et al.

5. Competition and Exchange Reactions: Probes for the DNA Binding Mechanism Katz’s model81 for binding of HgII to DNA emphasizes coordination of the electrophile to thymine,thymine (ThyH,ThyH) pairs to form Thy-Hg-Thy species with N3 proton displacement, in preference to other nucleoside pairs. CH3HgII has been shown17 to denature Ado,ThyH-rich DNAs in preference to GuoH,Cyd-rich ones. However, binding site preferences in these interactions are not clearly delineated because binding of HgII/CH3HgII to DNA and synthetic polynucleotides has not been understood with sufficient clarity at the molecular level. Competition and exchange reactions of HgII and CH3HgII with nucleic acid constituents, following the preparation of Hg-bridged complexes (Thy-Hg-Thy, 56, and Guo-Hg-Guo, 57) and CH3HgII complexes 21a and 58, have been investigated with the objective of streamlining information on the binding modalities of these Hg-based electrophiles.82,83

4.4. Other Amino Acids The interaction of CH3HgII with the amino acids glycine and alanine was studied by vibrational and X-ray spectroscopy.78 Complexes of 1:1 stoichiometry in which the metal is complexed via the amino function were obtained. Two different crystalline complexes were realized from aqueous mixtures of the dipeptide glycylglycine (GlyGly) and CH3HgII.79 In the 1:1 complex, a proton of the charged amino group is substituted by the metal ion. A 2:1 complex, 55, in which the second CH3HgII moiety is coordi-

nated to the carboxylate, is formed on prolonged treatment of GlyGly with a mixture of CH3HgOH and CH3HgClO4 in ethanol. A Raman spectroscopic investigation of several amino acids80 established the following general principles in the interaction of these biomolecules with CH3HgII: (i) All amino acids coordinate to CH3HgII via their terminal NH3+; facile coordination also occurs with -NH+ (histidine) and -NH (tryptophan). (ii) While terminal COO- coordination with the electrophile may be observed in the solid state, no binding with this function was detected in solution. (iii) Side-chain reactivities of NH3+ and COO- functions are lower than those of their terminal counterparts. (iv) Groups such as -OH (serine, threonine, and tyrosine), -NHC(NH2)2+ (arginine), and -C(dO)NH2 (asparagine and glutamine) show no reactivity toward CH3HgII coordination in aqueous solution. (v) The -SH group is the most reactive site in amino acids, being preferred to NH3+; substitution of H with CH3 renders the sulfur site unreactive toward CH3HgII in methionine, although some coordination may occur at very low pH ( N1-H (GuoH), although binding to both bases is significant. This conclusion provides direct support for Katz’s model81 for HgII binding to DNA, postulating initial reaction at appropriate ThyH,ThyH pairs which leads to the formation of Thy-Hg-Thy dimers. This initial process is followed by other cross-linking reactions, after all readily available ThyH,ThyH sites have been exhausted. It should however be pointed out that the mononucleoside models employed in this study are incapable of mirroring any stereochemical factors inherent in polymeric structures. This limitation was explored further in subsequent studies, as described below. Equilibration of GuoH with 56 or ThyH with 57 in pure DMSO83 in the ratio 2:1 results in identical, rapid, equilibrium redistribution of the species present in each system as revealed by in situ 1H and 13C NMR spectroscopic analyses. From each reaction system, the bridged complexes 56 and 57, uncomplexed nucleosides GuoH and ThyH, and the mixed bridged complex 59 were demonstrated85 to be present. Starting with GuoH + 56, or ThyH + 57, as reactants yielded the identical equilibrium composition of 56: 57:59, i.e., 3.2:1.5:1.0. Thus, the redistribution process establishes the relative thermodynamic stability order of 56 > 57 > 59. In a broader sense, these results support Katz’s chain slippage mechanism for HgII binding to DNA.81 The redistribution in DMSO could, in principle, occur via a three-centered exchange process or, in a secondary process, via a fourcentered transition state depicted as 60 and 61,

a Reprinted with permission from ref 82. Copyright 1985 Elsevier.

respectively.83 Precedents for such associative mechanisms have been proposed in other systems.43,86 A dissociative process (eqs 5, 15a, and 17) can also

Nuc2-H + Nuc1-HgOH h Nuc1-Hg-Nuc2 + H2O (9) where Nuc2-H is different in identity from Nuc1-H. Similar results were obtained82 in the exchange reactions involving ThyH and Guo-Hg-Guo in the ratio 2:1; the reaction sequence can be formulated as eqs 10-14.

ThyH + H2O h Thy- + H3O+

(10)

[Guo-Hg-Guo] + H3O+ h GuoH + Guo-Hg+ + H2O (11) Guo-Hg+ + Thy- h [Guo-Hg-Thy]

(12)

[Guo-Hg-Thy] + H3O+ h Thy-Hg+ + GuoH + H2O (13) Thy-Hg+ + Thy- h [Thy-Hg-Thy]

(14)

Of particular interest is the absence of the mixed bridged species Guo-Hg-Thy, 59. Since 59 is a plausible intermediate in the formation of 56 from the reaction of ThyH with 57 according to eqs 1012, it was concluded82 that 59 is a metastable species84 (see below for evidence for its formation). The exchange reaction involving CH3HgII was conducted by reacting molar equivalents of CH3HgGuo, 21a, and ThyH in water.82 Instantaneous exchange occurred; 1H and 13C NMR analyses of the reaction products in DMSO revealed extensive methylmercuration of ThyH and partial methylmercuration of GuoH. The equilibrium between the methylmercuriated and free nucleosides can be visualized as shown in Scheme 2 , for which eqs 5, 15, and 16


Onyido et al.

Table 1. 2J(1H-199Hg) Coupling Constants for Representative CH3HgII Complexes 2

J(1H-199Hg)/Hz

complex 20b [(CH3Hg)(InoH2)]NO3 21b [(CH3Hg)(InoH)] 22b [(CH3Hg)2(InoH)]NO3 25b [(CH3Hg)3(Ino)]NO3 30b [(CH3Hg)2(Th)]NO3 33 [{(CH3Hg)(Th)}2Hg](NO3)2 34a [(CH3Hg)(XanthH)] 35b [(CH3Hg)3(Xanth)]NO3 36 [(CH3Hg)4(Xanth)]NO3 37c [(CH3Hg)(ImH)] 39a [(CH3Hg)(MeImH)]NO3 39d [(CH3Hg)2(MeIm)]NO3 39e [(CH3Hg)(Me2Im)]NO3 46 [(CH3Hg)(MeImS)] 47a [(CH3Hg)(MeImSH)]NO3 47b [(CH3Hg)2(MeImS)]NO3 48a [(CH3Hg)(8-thioGuoH)] 48b [(CH3Hg)2(8-thioGuo)] 49a [(CH3Hg)(8-thioGuoH2)] 50a [(CH3Hg)(6-MPurH)] 50b [(CH3Hg)(2-A-6-MPurH)] 51c [(CH3Hg)3(6-MPur)]NO3 51d [(CH3Hg)3(2-A-6- MPur)]NO3

N-bound

S-bound

233.3 207.5 (211)c 221.2 215.8 227.4 232.2 211.8 219.1 213.4 196.0 222.1 214.2

C-bound

159.5

156.9 151.4 165.8 184.6 204.2d 207.8d 187.2 197.8d 211.9d 185.5 184.9 195.3d 193.1d

146.7 156.7

ref 34 34 31c 34 34 37a 37a 40 40 40 42a 42a 42a 42a 44 44 44 45 45 45 46 46 46 46

a The number of H atoms shown in each complex is equal to the number of potentially ionizable protons still present in the complex. b Abbreviations shown in the complexes are as follows for the basic skeletons of the ligands: Ino ) inosine; Th ) theophylline; Xanth ) xanthosine; Im ) imidazole; MeIm ) 1-methylimidazole; Me2Im ) 1,3-dimethylimidazolium ion; MeImS ) methimazole; 8-thioGuo ) 8-thioguanosine; 6-MPur ) 6-mercaptopurine riboside; 2-A-6-MPur ) 2-amino-6-mecaptopurine riboside. c Measured in D2O solution (see ref 31d). d Values are averaged coupling constants due to rapid CH3HgII exchange between N and S sites (see the text).

account for the results,43,83 the proton acceptor in this case being DMSO or adventitious H2O present in DMSO.

Nuc1-H + H2O h Nuc1- + H3O+

(5)

Nuc2-Hg-Nuc2 + H3O+ h Nuc2-Hg+ + Nuc2-H + H2O (15a) Nuc2-Hg+ + Nuc1- h Nuc2-Hg-Nuc1 (17) Circular dichroism (CD) studies of HgII-induced transitions in a series of polynucleotides87 support initial binding to thymine, in broad agreement with Katz’s model; this initial process, in turn, triggers duplex strand separation with concomitant cooperative binding to an adenine amino group of poly(dA) strands. Such studies on HgII-nucleic acid interactions also provide evidence for conformational changes,88 typically involving transitions from righthanded to left-handed structures.89 Recent NMR investigations of HgII binding to oligonucleotides,14,85 on the other hand, suggest that HgII binding to DNA is largely determined by the requirement for linear geometry at adenine N6 and thymine O4; thus, a cross-link is indeed formed with no consequence on the adenine-thymine N3-H‚‚‚N1 hydrogen bond or the H2‚‚‚O2 distance. Clearly, more work is needed to construct a model that fully correlates HgII effects with biomolecule complexity. In this regard, it is pertinent to point to a number of recent high-level quantum chemical studies90-92 which demonstrate qualitative differences between adenine-containing

and guanine-containing base pairs, with the consequence that the stabilities of adenine-thymine and guanine-cytosine base pairs are affected differently by interactions involving the hydrated metal ion.90,91 Thus, while the stability of adenine-thymine base pairing is enhanced through electrostatic effects, that of the guanine-cytosine pair is increased through a polarization mechanism.91

6. Diagnostic and Practical Utility of NMR Features of CH3HgII Complexes Certain 1H and 13C NMR features of some of the CH3HgII complexes presented above deserve some comment. 2J(1H-199Hg) coupling constants show significant sensitivity to the strength of the HgIIligand bond. In general, 13C chemical shifts are inherently more sensitive to the environment of the metal ion than are 1H chemical shifts; hence, the former provide a useful indicator of the nature of the donor ligand. For example, the conclusion that the ribose moiety of the nucleosides investigated is not involved in binding is reached on the basis that the sugar C atoms show only very slight changes in their 13 C chemical shifts upon complexation of the nucleobase with HgII/CH3HgII.33,34 One can generalize, from the 2J(1H-199Hg) values presented in Table 1 for representative examples, that coupling constants follow the order N- f S- f C-bound complexes. This order reflects the thermodynamic stability of the X-HgII bond (X ) N, S, or C donor atom) in which the C-HgII bond is the most stable and the N-HgII bond most labile; the S-HgII bond is of intermediate stability relative to the



Table 2. pKa Values at 25 °C for NH2 Groups of Several Nucleosides and Related Compounds Derived by Application of Eq 19a compound adenosine 9-methyladenine 1-methyladenosine guanosine 1,9-dimethylguanine

pKab,c 17.0 (18-19) 17.0 (16.7)e - (8.55)f 15.1 - (14.6)e

d

compound

pKab,c

1-methylguanosine cytidine 1-methylcytosine 2,3-O-benzylidene-5-O-tritylcytidine

14.9 15.5 - (16.7)e - (14.8)e

a Reprinted with permission from ref 26. Copyright 1981 Elsevier. b Estimated uncertainties (0.3 pKa unit. c Data in parentheses refer to literature values. d See: McConnell, B. Biochemistry 1974, 13, 4516. e See ref 94. f See: Hoo, D.-L.; McConnell, B. J. Am. Chem. Soc. 1979, 101, 7470.

C-HgII and N-HgII bonds. This thermodynamic order of stability differs from the observed ease of mercuration which manifests the hierarchy S- f Nf C-mercuration; C2/C8-mercuration has been shown above to be crucially dependent on protonation or metal coordination at N3/N7. 13C chemical shifts are considerably larger for S-bound complexes than for N-bound ones. In complexes containing S- and Nbound CH3HgII, the 13C signal is intermediate in value compared to those of the respective S-bound/ N-bound complexes, indicating rapid exchange of CH3HgII between the two centers (N and S) on the NMR time scale. 13 C NMR chemical shifts also elicit 1J(13C-199Hg) couplings which are significantly larger in magnitude than 2J(1H-199Hg) couplings and can be correlated with the latter according to eq 18. The origin of the relationship in eq 18 has been discussed.83 1

J ) 8.460(2J) - 155.6

(18)

The importance of the acidity of NH2 groups in defining complementarity of adenine-thymine and guanine-cytosine base pairing through H-bonding interactions underscores the need for reliable methods for the accurate measurement of their pKa values in the different nucleosides in which they occur. Data from our work33,34,42a,c and other laboratories43,60a,61,93 suggest that, for a given ligand donor atom, the magnitude of the coupling constant 2J(1H-199Hg), or simply J, of the CH3HgII complex is related to the basicity of the donor atom. We have found26,42a that eq 19 holds for sites that can undergo protona-

J ) -3.88pKa + 248.5

(19)

tion and methylmercuration, insofar as there is no 3-coordinate Hg in the CH3HgII complex. Application of eq 19 to J values measured for exocyclic NH2bound CH3HgII complexes provides the pKa values of the NH2 groups of several nucleosides and related compounds (Table 2), which compare well with literature values. The accessibility of NH2 pKa values, hitherto derived by indirect methods,94 through this simple NMR method enhances the prospects of quantifying the contributions of H-bonding95 and electronic complementarity96 to the specificity of base pairing. A number of interesting NMR studies, which reveal important structural details in the solution interactions of HgII with biomolecules, have appeared in the literature recently. 1H and 199Hg NMR spectroscopy was applied to the exchange reactions of the

complex 1,3-dimeU-C5-Hg-OOCCH3 (1,3-DimeU ) 1,3-dimethyluracil), 62, with a variety of anions and

model nucleobases.97 3J coupling of the 199Hg isotope with H6 of the 1,3-dimeU ligand was found to be diagnostic of the donor atoms trans to C5 of the uracil ring. 199Hg NMR spectra of mixed nucleobase complexes of the type 1,3-DimeU-C5-HgL (L ) second nucleobase) demonstrate a C-Hg-N arrangement, with strong binding of the electrophile to the second nucleobase at N3 of uracil, N3 of thymine, N4 of cytosine, or N1 of guanine. Binding to N7 of guanine was found to be weak. 199 Hg NMR studies and Biograf energy-minimum calculations of Hg2Cl2(cys,his-peptide) [cys,his ) Cys and His residues coordinated to HgII] show that the 199 Hg NMR chemical shift of the complexes Hg2Cl2(Z-cys-X-Y-his-OMe) (Z ) benzyloxycarbonyl; X-Y ) Ala-Ala, Ala-Pro, or Pro-Val) correlate with the coordinating ability of the Cys-X-Y-His moiety, which is a function of the interposed amino acid residues.98 The coordination modes of the cysteinethiolate groups are similar in the complexes. On the other hand, weak interactions occur between the histidine imidazole group and HgII; these interactions depend on the number of amino acid residues intervening between the Cys and His residues. Sensitivity of the 199 Hg NMR chemical shifts in the complexes to the steric effects of the amino acid residues was also demonstrated. The novel work of O’Halloran and co-workers99-101 provides direct structural data for MerR and MerRDNA adducts, and demonstrates the application of 199 Hg NMR spectroscopy to the study of the active sites of metalloproteins. Specifically, homonuclear and heteronuclear proton-detected 199Hg NMR data were employed to delineate the metal ion receptor environment of MerR and to characterize the allostery of MerR-DNA interactions by locating the ligands coordinating to HgII both in the MerR protein and in the protein-DNA complex. Comparison of the 199 Hg NMR chemical shifts of MerR and MerR-DNA adducts with those of model complexes and metalloproteins provides definitive information regarding the modes of HgII coordination.


7. Neurotoxicity of CH3HgII CH3HgII is a widespread and highly toxic environmental pollutant and has been long recognized as a neurotoxic hazard. Neuorological degeneration in animals and humans and the Minamata disease have been ascribed by medical scientists to high levels of ingested CH3HgII.102 There is, however, no agreement regarding the mechanism of its neurotoxicity103,104 as well as the relationship between the distribution of the toxicant in the central nervous system and CH3HgII neurotoxicity.105 It has been suggested that both CH3HgII accumulation and biotransformation are correlated with neurotoxicity105b and that the presence of CH3HgII affects the neuron in various ways that interfere with neurotransmission.106 In our study,107,108 the correlation between CH3HgII burden and its metabolism to HgII, and the structural damage in distinct regions of the mouse brain, was assessed following the administration of subchronic CH3HgII treatment. No correlation was found to exist between total HgII and structural damage, whereas CH3HgII was evenly distributed in the brain. However, a correlation was found between HgII concentration and the amount of structural damage observed in the anterior cerebral cortex. These observations suggest different mechanisms of sensitivity to HgII in the different areas of the brain and emphasize a possible role for the inorganic metabolite of CH3HgII in the anterior cerebral cortex.109 The distribution of inorganic HgII, presumably resulting from demethylation of CH3HgII, and the disruption of the blood-brain barrier have been demonstrated in the central nervous system of rats using autometallographic techniques.109 Results from studies on the immunomodulating effects of CH3HgII on mice110 and of HgII and CH3HgII on CaII fluxes in rat brain microsomes111 have appeared recently. CH3HgII has been reported to increase cystolic CaII concentration in rat cerebrum synaptosomes112 as well as inhibit ATP synthesis.113

8. Mercury Detoxification Strategies Mercury bioaccumulates as CH3HgII in the food chain. Its dynamic redox chemistry in the atmosphere and condensation via climatic mechanisms lead to widespread contamination of soils and water. It is therefore an important concern that environmental mercury pollution and associated effects be kept to the barest minimum. This calls for the development of effective technologies for reducing the mercury burden of the environment and for treating environments that are already mercury-contaminated. In this section, various emerging methods and technologies for remediation of mercury pollution and intoxication are reviewed.

8.1. Anthropogenic Methods The conventional methods for removal of mercury from contaminated sites are mainly physical or physicochemical, such as dredging and landfilling of hazardous sediments, precipitation, chemical coagulation, or adsorption.114 These methods either are

Onyido et al.

costly or generate secondary pollution.115 This has led to the search for new, convenient, safe, and possibly more cost effective methods of environmental remediation. A recent study116 demonstrated that CH3HgCl is degraded by HO• radicals which are generated via nitrate photolysis in the wavelength range of 285800 nm with a 450 W xenon lamp. HgII, Hg0, CHCl3, and CH2O were identified as products of the process. It is argued that formation of formaldehyde as one of the reaction products is good evidence that the reaction proceeds by way of C-Hg bond fission. The second-order rate constant (9.83 × 109 M-1s-1) calculated for the process at pH 5 and room temperature with benzoic acid as scavenger enabled an estimation of the CH3HgII degradation rate in natural waters, leading to the conclusion that HO• degradation of organomercurials may be one of the important natural pathways in surface water for reducing the levels of this toxicant in the environment (see below). Electrokinetic means for in situ remediation of contaminated soils, in which an imposed electric field affects the directional migration of the contaminant, have been suggested,117 and laboratory-scale experiments have been published.118 The potential for this method was demonstrated recently for the removal of ionic mercury from contaminated soils.119 It was shown that HgII is mobilized toward the anode, probably as HgI42-. A variant of this principle120 involves the addition of I2/I-, as a lixiviant near the cathode, to contaminated soils. The presence of the lixiviant ensured the oxidation of reduced mercury in the soil and its complexation and subsequent transport as the complex ion HgI42-. A method which utilizes acidic KI to clean up mercury-contaminated soils in the absence of an applied electric field has also been demonstrated on the bench scale.121 Repeated passage of acidic KI solution (pH 1.5) through a column packed with the contaminated soil (containing 47.1 mg of Hg/g) decreased the mercury content by ca. 76%. The HgI42in the leachate from the column was then treated with activated carbon. Removal of inorganic mercury contaminant from wastewaters using a crandalitte-type compound with the formula Ca0.5Sr0.5Al3(OH)6(HPO4)(PO4) has been described.122 CaII and SrII exchanged with HgII in the wastewater according to eq 20, reducing the mercury content from 90 to [ThyHg-Guo]. Characterization of the mixed bridged species was achieved in DMSO.


Recent CD data argue for conformational changes induced by HgII binding to DNA, involving transitions from right-handed to left-handed structures. NMR studies on oligonucleotides suggest that HgII binding to DNA is determined by the necessity to maintain linear geometry at adenine N6 and thymine O4. Models capable of correlating the specific nature of CH3HgII/HgII-biomolecule interactions with progressive biomolecule complexity are required for a thorough understanding of the structural consequences of HgII binding to DNA at the molecular level. Crystal structures for a number of complexes provide direct evidence for binding sites and demonstrate H-bonding and secondary bonding interactions involving Hg in the solid state. A possible role for the inorganic metabolite of CH3HgII in neurotoxicity is suggested. Nature’s strategies for mercury detoxification, as exemplified by some bacteria, underscore the huge potential in deploying these strategies in a rational manner for environmental remediation, reversal of mercury intoxication in humans, and the exploration of chemical mimics of these strategies already optimized by nature. Recent efforts at genetic engineering adaptations of the bacterial methods of mercury detoxification demonstrate promise for everyday application.

10. Acknowledgments We are greatly indebted to the numerous coworkers and collaborators whose dedicated work made possible the research presented in this review. We thank the reviewers for constructive critical comments and helpful suggestions. Funding support by the Natural Sciences and Engineering Research Council of Canada (E.B., A.R.N.) and the Canadian International Development Agency (I.O.) is also gratefully acknowledged.

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